Hepatitis B is a life-threatening disease that affects millions of individuals worldwide. Several vaccines are currently available to prevent initial infection with the hepatitis B virus (HBV). However, many patients become chronically infected with the virus, becoming potential sources of spread of the disease, and suffering the high risk of liver ailments associated with long-term infection, such as chronic hepatitis, cirrhosis and hepatocellular carcinoma.
Chronic hepadnavirus infection is extremely difficult to eradicate for several reasons: (1) chronically infected patients develop immunotolerance to vital surface antigens; (2) the hepatitis B virus does not kill host hepatocytes, so vital infection cannot be overcome simply by preventing new infection and allowing already-infected host cells to die; (3) the covalently closed circular form of the hepadnavirus genome does not self-replicate and is therefore unaffected by agents which inhibit DNA-directed DNA synthesis; (4) hepatitis B virus exists in non-hepatic tissues, so reinfection of hepatic tissue may result from virus located in other tissues; and (5) the hepadnavirus genome can integrate into hepatocyte chromosomes, and thus exist in a dormant, non-replicative form for many years. See P. L. Marion, Current Topics in Microbiology and Immunology, Vol. 168: 167-183 (1991). In spite of these obstacles, certain steps in the replication cycle of hepadnaviruses have been cited as potential targets for anti-viral therapy. One potential target is the replication of the hepadnavirus genome by reverse transcription of the RNA pregenome. This step is essential in the life cycle of the virus.
Reverse transcription of the RNA pregenome is catalyzed by a gene product encoded by the hepadnavirus pol gene. The pol gene encodes three activites: (1) DNA priming activity; (2) reverse transcriptase (i.e., RNA-directed DNA polymerase) activity; and (3) RNAse H activity. It has heretofore not been known whether these three catalytic functions are embodied in one or several separate gene products. In fact, little is known about the product or products of the viral pol gene, and this lack of knowledge has frustrated attempts to develop anti-viral agents targeted to the pol gene product.
Efforts to obtain more detailed information about the mechanism and biochemistry of the reactions directing reverse transcription of the hepadnavirus genome have been stymied by the inability to obtain enzymatically active hepadnavirus polymerase gene product. See, e.g., Bayand et al., J. Virol., 63: 1019-21 (1989). Consequently, there has been no feasible means to study or screen for anti-viral agents directed toward this protein or set of proteins. Thus, a potentially useful target for anti-viral therapy has remained virtually unexplored. A need clearly exists for the production of a purified hepadnavirus polymerase gene product that can be used for in vitro assays to screen for potential anti-viral agents. A concomitant need exists for methods to measure the various activities of such a gene product, once it becomes available.